PROJECT SUMMARY Legionella pneumophila is an opportunistic bacterial pathogen that causes outbreaks of a lethal, pneumonia- like disease. Although human infections are evolutionary ?dead ends? for these bacteria, Legionella nevertheless carry extensive molecular arsenals to attack human cells due to their adaptation to their natural hosts, environmental amoebae. Residence in amoebae also protects Legionella from antibiotics and other efforts to eliminate the bacteria from man-made structures, perpetuating human outbreaks. Revealing how Legionella exploits host amoebae?particularly steps vulnerable to disruption?therefore has direct benefits for human health. This proposal will investigate how molecular ?arms races? between Legionella and amoebae have shaped the molecular toolkit of this pathogen, and address the specific hypothesis that Legionella secreted effector proteins are engaged in arms races with amoeba immune pathways. The experimental tractability and evolutionary resources available for Legionella and amoebae make this a powerful host- microbe model system, where it is possible to test the functional consequences of evolutionary innovation in both host and microbe. Aim 1 will investigate how Legionella has been impacted by such an arms race. Evolutionary approaches will analyze this organism's enormous arsenal of molecular weaponry, the type IV effectors, to identify genes and residues likely engaged in arms races with hosts. Bacterial genetics will then be used to functionally test evolutionary hypotheses about which genes or residues are critical for pathogen fitness. These studies will begin with the mavN gene, which appears to be engaged in an evolutionary ?battle for iron? within host cells. In addition to such competitions for resources, many pathogens have evolved strategies to evade detection by host immune systems. High-throughput transposon-sequencing approaches will be used to identify bacterial genes that are required for fitness within Dictyostelium amoebae, particularly those that interact with amoeba immunity. These experiments will reveal which Legionella proteins have experienced strong selective pressures in amoeba hosts. Aim 2 will examine the host genes likely to place strong selective pressures on Legionella through studies of amoeba immune defenses. The Dictyostelium TirA protein is related to Toll-like receptors in animals, and helps the amoebae to resist Legionella infection. However, beyond these basic facts, almost nothing is known about amoeba immunity. This aim will further characterize the activity of the TirA immune pathway, identifying additional members of the immune pathway and transcriptional targets. Evolutionary and unbiased genetic approaches will highlight additional arms of the amoeba immune response that respond to Legionella infection. The proposal will combine the applicant's background in evolution, genetics, and host-microbe interactions with the Malik lab's expertise in evolutionary arms races. This interdisciplinary approach will also provide her new training in bacteriology and amoebal biology, to uncover how evolutionary arms races in the natural environment have armed Legionella for human infections.